Peptide nucleic acid (PNA) is a DNA/RNA mimic that recognizes complementary sequences by Watson-Crick base-pairing and binds to RNA with high affinity. We have developed efficient methods for synthesizing PNAs and introducing them into cells. Once inside cells, we have shown that PNAs can efficiently inhibit gene expression. The long-term objective of this proposal is to better understand the properties of antisense PNAs and test the hypothesis that PNAs can be useful agents for probing biological function inside cells. Specifically, we propose to 1) Characterize the rules governing successful use of antisense PNAs to inhibit gene expression, 2) Use antisense PNAs to examine the consequences of inhibiting significant biological targets, thereby demonstrating that PNAs can be useful tools for studying cellular phenotypes, and 3) Use PNAs to detect gene expression inside cells. To achieve these goals my laboratory will take advantage of our ability to rapidly synthesize PNAs and PNA-peptide conjugates and our experience with using PNAs inside cells. The potential for compounds that recognize nucleic acids to treat disease and be powerful research tools is enormous. Few, if any, other approaches to drug discovery can move through the initial stages of development so rapidly. In spite of compelling advantages, progress in the clinic has been slow. Antisense PNAs possess dramatically different chemical properties relative to RNA or DNA oligomers that can also block gene expression. These chemical differences may confer advantages upon PNA and encourage the development of new research tools or more effective therapeutics. The work proposed here will rigorously test the value of antisense PNAs and our data will shape decisions regarding the use of PNAs for laboratory studies and clinical development.